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Monday, October 27, 2014

Synthetic Genome Assembly in Your Research Lab

The Gibson Assembly® method was first developed by Dr. Dan Gibson and his colleagues at the J. Craig Venter Institute. With Dr. Gibson’s expertise, they successfully synthesized a 583kb Mycoplasma genitalium genome in 2008 and a 1.2 million bp Mycoplasma mycoides genome in 2010, the largest synthetic genome to date. The team overcame the limitations of traditional cloning by developing the Gibson Assembly® method, which allows for seamless DNA assembly without the presence of restriction enzyme sites. Since the likelihood of unwanted restriction sites increases with the length of the sequence, large genomic sequences could not be assembled prior to this method. Assembly in Stages

The Gibson Assembly® reaction relies on a mix of enzymes (exonuclease, polymerase, and ligase) that anneal overlapping ends between DNA fragments in an isothermal reaction. The team built the synthetic genome in several stages: 1000 bp cassettes were assembled from overlapping synthetic oligos, which were recombined in sets of 10 to produce 10,000 bp assemblies. The 10kb assemblies were further recombined to produce 100kb assemblies. From there, these fragments formed the complete 1.2 million base pair genome.

Dr. Gibson joined Synthetic Genomics Inc. in 2011 after more than 6 years in JCVI. He brought the synthetic genomic tools developed at JCVI to SGI, leading to the development of automated DNA synthesis, assembly, and error correction technology used today at SGI-DNA. 1 Step Away From a Perfect Clone

The Gibson Assembly® method has been developed into a high fidelity 1 step reagent kit by Dan Gibson’s team at Synthetic Genomics Inc. SGI’s Gibson Assembly® HiFi 1 Step Kit is now available for purchase through SGI-DNA. Vibhu Gupta, Senior Product Manager at SGI, says "the main advantage of the new formulation compared to other commercial kits is its ability to accurately assemble multiple fragments with greater than 90% cloning efficiency." The team's experimental results show that the high fidelity formulation ensures assembly junctions are error free, resulting in more perfect clones and lower sequencing costs. The kit is also capable of assembling large genome sized fragments up to 100kb. Commercial licensing is now available to companies interested in assembling synthetic plasmids, pathways and genomes that result in commercial products.

SGI-DNA uses the Gibson Assembly® method today to assemble large and complex DNA constructs, delivering synthetic genomes and pathways to its commercial customers. The Gibson Assembly® technology will be prominently featured in SGI’s future product launches. Stay tuned for more news.